Investigations will be continued on the inaccessible subcellular compartment underlying quantal transmitter release at motor nerve terminals. This will involve a new methodology, specifically developed to answer criticisms of the previous technique. Significant advances provided by this approach are the ability to (a) obtain unbiased estimates of the quantal release parameters m, n, and p, (b) calculate variance of p (varsp) which can be used to monitor real-time changes in [Ca2+]i distribution, and (c) utilize miniature potentials (mepps) alone to compute release parameters (rather than mepps and nerve-evoked potentials). This should allow studies of subcellular processes and drug actions which were not previously possible. Among these will be the: (1) effect of [Ca2+]o on release parameters (to provide data for the case of a uniform [Ca2+]i distribution at transmitter release sites); (2) effect of Sr2+ on varsp and n and p, to see if it acts at intracellular organelles or at release sites, and to compare the efficacy of Sr2+ vs. Ca2+ at transmitter release sites, by comparing the n and p values which produce equal values of m; (3) effect of temperature on each release parameter--if it reflects a biochemical (high Q10) or a biophysical (low Q10) process (results to be compared to a theoretical model); (4) effect of hyperosmotic pressure on release parameters, using non-permeating (sucrose) as well as permeating (alcohol) agents; (5) effect on release parameters of uptake of acetylcholinesterase or horseradish peroxidase into synaptic vesicles (effect of hemicholinium-3 on the cytoplasmic pool of ACh and on vesicle loading will also be tested); (6) effects of specific pharmacologic or toxicologic agents, e.g., methylmercury; others may include tetraethyllead, Al3+, Cu2+ and heavy metal chelators; (7) effect of mitochondrial and Na pump inhibitors on varsp (to distinguish direct vs. indirect actions of Na+ to promote transmitter release); (8) computation of release parameters for mepps and sub-mepps recorded at the same junction, to see if mepps are comprised of sub-mepps; (9) to examine the Ca dependence (cooperativity) of n, m, p and varsp before and after BoTX poisoning to elucidate the mechanism of toxicity. Results of these physiologic studies should provide insight into the mechanisms involved in Ca2+ regulation at the subcellular level and document the usefulness of the new method for carrying out subsequent investigations.